Human mitochondrial proteins and polynucleotides encoding the proteins

ABSTRACT

The invention of the present application provides an isolated and purified human mitochondrial protein comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, which is a novel human protein promoting aggregation and fusion of mitochondria. The present invention also provides a polynucleotide encoding such a mitochondrial protein, antibody against such a mitochondrial protein, and a proteoliposome composed of such a mitochondrial protein and lipid. Mitochondrial proteins are useful for clarifying causes of mitochondrial diseases as well as for developing preventive and therapeutic methods thereof. Furthermore, antibodies and probes derived from genes encoding such proteins are potentially useful materials for diagnosis of condition of mitochondria in particular diseases. Furthermore, proteoliposomes provide measures for specific transfer of foreign genes or drugs targeted toward mitochondria.

This application is a U.S. national stage of PCT/JP01/09783 filed Nov.8, 2001.

TECHNICAL FIELD

The invention of the present application relates to proteins derivedfrom human mitochondria, polynucleotide encoding such proteins, andantibodies against such proteins. More specifically, the invention ofthe present application relates to NOVEL HUMAN PROTEINS AND ANTIBODIESwhich are useful for diagnosis and treatment of a variety of diseasescaused by mutation of mitochondrial DNA. The invention also relates toPOLYNUCLEOTIDES which are useful as probes for gene diagnosis of theabove diseases, gene sources for gene therapy, and gene sources for massproduction of the proteins according to the present invention. Theinvention further relates to LIPOSOMES (LIPOSOME VECTORS) which enablemitochondria-specific transfer of foreign genes or drugs.

BACKGROUND ART

Mitochondria are organelles each composed of an outer membrane and aninner membrane having a cristae structure, and are distributedthroughout cytoplasm in a tubular reticular structure. Mitochondria alsohave their own genes (mitochondrial DNA) besides nuclear genes.

Mitochondria have the functions of, for example, producing energy whichis necessary for activity of cells, and catalyzing biosynthesis anddegradation of crucial biological substances. Mitochondria are alsoinvolved in other biological activities such as production of activeoxygen and production of apoptosis-inducing signal.

Mitochondria dynamically change their forms by migration, fusion anddisintegration in response to environmental changes in the cell. Inparticular, under the pathological conditions, such as the case of liverdisease, congenital muscular dystrophy, gastric cancer, myeloma, anddilated cardiomyopathy due to abnormality of mitochondrial DNA,mitochondria significantly change their forms and distributions, andexpress megamitochondria, annular or axle form, or morphological andstructural variations having an annular or concentric cristae structure.

As a mammalian gene in relation to these mitochondria, a gene involvedin disintegration of mitochondria is known (J. Cell Biol. 143: 351–358,1998). On the other hand, as a gene involved in fusion of mitochondria,a gene product Fzo expressed at the time of formation of sperm ofdrosophila, which promotes mitochondrial fusion (Cell 90: 121–129,1997), and a gene product Fzo1p which promotes fusion of mitochondriaoccurring at the time of meiotic division of fission yeast (J. CellBiol. 143: 359–373, 1998) are known. However, mammalian genes involvedin fusion of mitochondria have not been identified.

Examples of the diseases caused by mutation of mitochondrial DNA includemitochondrial myopathy, cardiomyopathy, type II diabetes, Alzheimer'sdisease, Parkinson's disease and the like. Such a mutant mitochondrialDNA exists within a cell in the state of heteroplasmy wherein mutantmitochondrial DNA and normal mitochondrial DNA coexist. When theexisting ratio of mutant DNA exceeds a predetermined threshold, the cellfunction deteriorates to lead appearance of disease symptoms. Genetherapeutic methods against these diseases have not been establishedbecause, unlike nuclear DNA, mitochondrial DNA exists in mitochondria.As a method which enables gene therapy targeted towards mitochondrialDNA, transfers of foreign mitochondria by means of cybrid method ormicroinjection (J. Cell Biol. 67: 174–188, 1975, Cell 52: 811–819, 1988)have been reported heretofore. However, these methods entail thedrawbacks that large amounts of cytoplasm components other thanmitochondria are introduced, and that desired genes and substancescannot be introduced into mitochondria. Therefore, they are far fromactual use at present.

As described above, mitochondria changes their forms in accordance withthe pathological condition of particular disease, and a proteinpromoting aggregation and fusion of mitochondria is thought to beinvolved in such a morphological change. Therefore, it is expected thatisolation of human proteins as described above and detailed analysis oftheir functions will open new avenues for clarifying causes ofmitochondrial diseases in human and developing preventive andtherapeutic methods thereof. Moreover, it is expected that antibodiesagainst such proteins and probes derived from genes encoding suchproteins will be useful materials for diagnosing conditions ofmitochondria in a particular disease.

Furthermore, protein samples which promote aggregation or fusion ofmitochondria are expected to provide new measures for specific transferof foreign genes and drugs targeted on mitochondria.

DISCLOSURE OF INVENTION

It is an object of the invention of the present application to providenovel human proteins which promote aggregation or fusion ofmitochondria, and polynucleotides encoding such proteins.

It is also an object of the invention of the present application toprovide antibodies against the proteins, and proteoliposomes whichcontain such protein as an active ingredient.

In order to achieve the above objects, the present application providesthe following inventions (1) to (8).

-   (1) An isolated and purified human mitochondrial protein comprising    the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4.-   (2) A polynucleotide encoding the protein of the invention (1).-   (3) The polynucleotide of the invention (2), which comprises the    base sequence for the protein translation region in SEQ ID NO: 1 or    SEQ ID NO: 3.-   (4) A polynucleotide with which a polynucleotide having the sequence    of SEQ ID NO: 1 or SEQ ID NO: 3 or a part thereof hybridizes under a    stringent condition.-   (5) An expression vector, which expresses the polynucleotide of the    invention (3) in the in vitro translating system or in a host cell.-   (6) A transformant with the expression vector of the invention (5),    which produces the human mitochondrial protein of the invention (1).-   (7) An antibody against the human mitochondrial protein of the    invention (1).-   (8) A proteoliposome composed of the human mitochondrial protein of    the invention (1) and lipid.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a photographic image taken with a confocal laser scanningmicroscope, showing the condition of mitochondria stained with rhodamine123 in a HeLa cell into which cDNA of Hfzo genes has been introduced.

FIG. 2 is a photographic image taken with a confocal laser scanningmicroscope, showing the mitochondria and localization of Hfzo-GFP fusionproteins in a HeLa cell into which pDs Red1-Mito gene encoding redfluorescent protein having a mitochondrial target signal and Hfzo-GFPfusion genes have been introduced.

BEST MODE FOR CARRYING OUT THE INVENTION

The protein according to the invention (1) refers to two kinds ofisolated and purified human proteins respectively having amino acidsequences of SEQ ID NO: 2 and SEQ ID NO: 4 (hereinafter, referred to as“Hfzo1 protein” and “Hfzo2 protein”, respectively, or “Hfzo proteins”for both). Each of these proteins coded by nuclear genome is expressedat nucleus, migrates toward mitochondrial membrane to localize there,and thereby functions to promote aggregation and induction ofmitochondria. To be more specific, these proteins localized at differentmitochondrial membranes are bound with each other, whereby aggregationand fusion among mitochondria are achieved. These proteins are theproteins that are first characterized in mammal. These proteins havecoiled-coiled domains, a GTPase domain and transmembrane domains intheir primary structure, so that similarity with the primary structureof a device (SNARE, rab, syntaxin or the like) involved in fusion ofvesicle is found.

These Hfzo proteins according to the invention (1) can be obtained, forexample, by isolating from mitochondria of human cell, by preparing apeptide by way of chemical synthesis based the amino acid sequence ofSEQ ID NO: 2 or SEQ ID NO: 4, or by producing by recombinant DNAtechnique using a polynucleotide encoding the amino acid sequence of SEQID NO: 2 or SEQ ID NO: 4. Among these, the method based on therecombinant DNA technique is preferably used. For example, by preparingan RNA from a vector harboring the polynucleotide (ORF region of cDNA)according to the invention (3) by in vitro transcription, followed by invitro translation with the use of this RNA as a template, it is possibleto achieve in vitro expression of protein. In addition, by recombiningthe polynucleotide into an appropriate expression vector using knownmethods, it is possible to express the protein encoded by thepolynucleotide on a large scale in prokaryotic cells such as Escherichiacoli and Bacillus subtilis, or in eukaryotic cells such as yeast, insectcells and mammalian cells.

In the case of expressing DNA by in vitro translation and producing theHfzo protein according to the invention (1), the polynucleotideaccording to the invention (3) is inserted into a vector having an RNApolymerase promoter to construct a recombinant vector (invention (5)).Then this vector is added to an in vitro translation system such asrabbit reticulocyte lysate or wheat germ extract containing an RNApolymerase corresponding to the promoter, whereby the protein accordingto the invention (1) is produced in vitro. Examples of the RNApolymerase promoter include T7, T3 and SP6. Examples of vectorscontaining these RNA polymerase promoters include pCMV-SPORT, pKA1,pCDM8, pT3/T7 18, pT7/3 19 and pBluescript II.

In the case of expressing DNA in a microorganism such as Escherichiacoli to produce the Hfzo protein according to the invention (1), thepolynucleotide according to the invention (3) is recombined into anexpression vector which is replicable in a microorganism and having anorigin, promoter, ribosome binding site, DNA cloning site, terminatorand the like, to thereby create an expression vector (invention (5)).Then a host cell is transformed with this expression vector, and theresultant transformant (invention (6)) is cultured. In this manner, aprotein encoded by the present polynucleotide can be produced in themicroorganism on a large scale. In this case, by expressing DNA in whichan initiation codon and a stop codon are added before and after a giventranslation region, it is possible to obtain a protein fragmentincluding the given region. Alternatively, it may be expressed in theform of a fusion protein with other protein. By cutting this fusionprotein with an appropriate protease, it is possible to acquire only aprotein portion encoded by the present polynucleotide. Examples ofexpression vector to be used for Escherichia coli include pUC series,pBluescript II, pET expression system, pGEX expression system, pQEexpression system and the like.

In the case of expressing DNA in an eukaryotic cell to produce the Hfzoprotein according to the invention (1), the polynucleotide according tothe invention (3) is inserted into an expression vector for eukaryoticcell having a promoter, splicing regions, poly(A) addition site and thelike to create a recombinant vector (invention (5)), and then therecombinant vector is introduced into an eukaryotic cell (invention(6)). In this manner the protein according to the invention (1) can beproduced in a eukaryotic cell. Examples of the expression vector includepKA1, pCDM8, pSVK3, pMSG, pSVL, pBKCMV, pBK-RSV, EBV vector, pRS, pYES2and the like. By using pIND/V5-His, pFLAG-CMV-2, pEGFP-N1, pEGFP-C1 andthe like as an expression vector, it is possible to express fusionproteins to which various tags such as His tag, FLAG tag, GFP and thelike are added. As the eukaryotic cell, mammalian culture cells such assimian kidney cell COS7 and Chinese hamster ovarian cell CHO,Saccharomyces cerevisiae, Schizosaccharomyces pombe, silkworm cell,platanna egg cell are commonly used, however, any eukaryotic cells canbe used insofar as they can express the protein according to theinvention (1). For introducing the expression vector into the eukaryoticcell, known methods such as electroporation, calcium phosphate method,liposome method and DEAE-dextran method can be used.

For isolating and purifying an objective protein from the culturefollowing expression the Hfzo protein according to the invention (1) ina prokaryotic or eukaryotic cell, known separating operations are usedin combination. Examples of such operations include treatment withdenaturant such as urea or with surfactant, ultrasonic treatment, enzymedigestion, salt precipitation or solvent precipitation method, dialysis,centrifugation, ultrafiltration, gel filtration, SDS-PAGE, isoelectricfocusing, ion-exchange chromatography, hydrophobicity chromatography,affinity chromatography, reverse-phase chromatography and the like.

The Hfzo protein according to the invention (1) implies peptidefragments (of 5 or more amino acid resides) comprising any partial aminoacid sequences of the amino acid sequence represented by SEQ ID NO: 2 orSEQ ID NO: 4. These peptide fragments can be used as antigens forpreparing antibodies. Furthermore, the protein according to theinvention (1) implies fusion proteins with other arbitrary proteins. Forexample, fusion proteins with glutathione-S-transferase (GST) or greenfluorescent protein (GFP) can be exemplified. Furthermore, the proteinaccording to the invention (1) possibly undergoes various modificationsin a cell after translation. Therefore, proteins resulting from suchmodifications also fall within the bound of the protein according to theinvention (1). Examples of such post-translational modification includeleaving of N-terminal methionine, N-terminal acetylation, addition ofsugar chain, restrictive lysis by intracellular protease,myristoylation, isoprenylation, phosphorylation, and the like.

The invention (2) provides a polynucleotide encoding the protein of theinvention (1), and implies human nuclear DNA (genomic DNA), mRNA andcDNA thereof (concretely, polynucleotides having base sequencesrepresented by SEQ ID NO: 1 and SEQ ID NO: 3, respectively), andcomplementary strands thereof.

The polynucleotide of the invention (3) refers to two kinds of cDNAscomprising a base sequence which constitutes the translation region(open reading frame: ORF) of SEQ ID NO: 1 or SEQ ID NO: 3. Since theprotein of the invention (1) is expressed in any cells, by screeninghuman cDNA library constructed from human cells with the use ofoligonucleotide probes synthesized on the basis of the base sequence ofSEQ ID NO: 1 or SEQ ID NO: 3, it is possible to readily acquire the sameclone as the polynucleotide of the invention (3). cDNA is synthesized byusing poly(A)+RNA extracted from human cells as a template. The humancells may be those surgically removed from human body or culture cells.cDNA can be synthesized by using known methods (Okayama, H. and Berg,P., (1982) Mol. Cell Biol. 2, 161–170; Gubler, U. and Hoffman, (1983) J.Gene 25, 263–269; Kato, S. et al. (1994) Gene, 150, 243–250).Alternatively, objective cDNA may be synthesized by an RT-PCR methodusing these poligonucleotides as primers and an mRNA isolated from humancell as a template.

In general, polymorphism due to individual specificity is often observedin human genes. Therefore, polynucleotides wherein addition or deletionof one or more nucleotide(s) and/or substitution with othernucleotide(s) is made in the base sequence of SEQ ID NO: 1 or SEQ ID NO:3 also fall within the bound of the polynucleotide according to theinvention (3).

Similarly, proteins wherein addition or deletion of one or more aminoacid(s) generated due to these alteration and/or substitution with otheramino acid(s) is made also fall within the bound of the proteinaccording to the invention (1) insofar as the have Hfzo proteinactivity.

Furthermore, the polynucleotide according to the invention (3) impliesDNA fragments (10 bp or more) comprising any partial base sequences ofthe base sequence of SEQ ID NO: 1 or SEQ ID NO: 3. Also, DNA fragmentsmade up of a sense strand and an antisense strand fall within thisbound. These DNA fragments can be used as probes for gene diagnosis.

The invention (4) provides a polynucleotide to which a polynucleotidecomprising a sequence represented by SEQ ID NO: 1 or SEQ ID NO: 3 or apartial continuous sequence thereof will hybridize in a stringentcondition, and implies partial regions of nuclear DNA of all mammalsincluding human (DNA fragments), as well as mRNAs and cDNAs thereof.Herein, the term “stringent condition” refers to a condition that allowsselective and detectable specific connection between a polynucleotidecomprising a base sequence of SEQ ID NO: 1 or SEQ ID NO: 3 or a partialcontinuous sequence thereof (for example 10 bp or more) and nuclear DNA.The stringent condition is defined by salt concentration, organicsolvent (for example, formamide), temperature and other knownconditions. That is, stringency is increased by reducing the saltconcentration, by increasing the concentration or organic solvent or byelevating the hybridization temperature. For example, the stringent saltconcentration is usually about 750 mM or less of NaCl and about 75 mM orless of sodium triphosphate, more preferably about 500 mM or less ofNaCl and about 50 mM or less of sodium triphosphate, and most preferablyabout 250 mM or less of NaCl and about 25 mM or less of sodiumtriphosphate. The stringent concentration of organic solvent is about35% or more, and most preferably about 50% or more in the case offormamide. The stringent temperature condition is about 30° C. or more,more preferably about 37° C. or more, and most preferably about 42° C.or more. Examples of other conditions include hybridization time,concentration of washing agent (for example, SDS) and presence/absenceof carrier DNA, and combinations of these conditions will providevarious stringencies. Also the condition of washing after hybridizationwill influence on the stringency. This washing condition is also definedby salt concentration and temperature, the less the salt concentrationand the higher the temperature, the more the stringency of washing. Forexample, the stringent salt condition for washing is preferably about 30mM or less of NaCl and about 3 mM or less of sodium triphosphate, andmost preferably about 15 mM or less of NaCl and about 1.5 mM or less ofsodium triphosphate. The stringent temperature condition for washing isabout 25° C. or more, more preferably about 42° C. or more, and mostpreferably about 68° C. or more. The mitochondrial DNA fragmentaccording to the invention (4) can be isolated by screening a genomelibrary prepared from human nuclear DNA through hybridization andwashing treatment under stringent conditions as described above usingthe aforementioned polynucleotide as a probe.

The polynucleotide (genomic DNA fragment) of the present invention (4)includes expression regulating regions (promoter/enhancer, suppressersequences or the like) with respect to the Hfzo protein coding region.These expression regulating sequences are useful as materials forscreening substances which regulate in vivo expression of the Hfzoprotein.

The antibody according to the invention (7) can be obtained from serumof an animal which has been immunized by using the Hfzo protein of theinvention (1) as an antigen. As the antigen, peptides that arechemically synthesized based on the amino acid sequence represented bySEQ ID NO: 2 or SEQ ID NO: 4, Hfzo proteins that are expressed ineukaryotic cells or prokaryotic cells can be used. Alternatively, theantibody may be prepared by sampling serum after introducing theaforementioned expression vector for eukaryotic cell into muscle or skinof animal by injection or gene gun (for example, invention disclosed inJapanese Patent Application Publication (JP-A) No. 7-313187). As theanimal, mouse, rat, rabbit, goat, chicken and the like can be used. Bypreparing a hybridoma by fusing a B cell sampled from a spleen ofimmunized animal with a myeloma, it is possible to produce a monoclonalantibody against the Hfzo protein.

The invention (8) provides a proteoliposome composed of the Hfzo proteinof the invention (1) and lipid, and can be used as a liposome vectorwhich encapsulates foreign genes or drugs so as to achieve mitochondrialspecific gene transfer or drug administration. In brief, inventors ofthe present invention created proteoliposome using a membrane fractionof mouse hepatocyte mitochondria and lipid, encapsulated a fluorescentpigment marker in the proteoliposome, and microinjected theproteoliposome into a mouse fertilized egg, and finally found that thelocalization of inherent mitochondria coincides with the localization ofliposome (Biochem, Biophys, Res. Comm. 278: 183–191, 2000). Thissuggests that mitochondrial membrane protein constituting the liposomerecognizes the same membrane protein of inherent mitochondrial and bindthereto, thereby causing aggregation and fusion of liposome and inherentmitochondria.

Such a proteoliposome can be constructed by preparing a liposome in aknown manner, and mixing the aforementioned Hfzo protein according tothe invention (1) with this liposome, followed by freezing, melting andthe like operations.

EXAMPLES

Now the present invention will be explained more concisely andconcretely by way of examples, however, it is to be noted that thepresent invention is not limited to these examples.

Example 1 Cloning of cDNA

A human EST library was searched on the basis of the base sequence ofcDNA of drosophila Fzo gene (Cell 90: 121–129, 1997; GenBank AccessionNo. U95821), and using highly homologous ESTs as probes, a cDNA libraryderived from human heart was searched. As a result of this, two positiveclones were obtained. 5′ side and 3′ side were synthesized in accordancewith RACE (Rapid amplification of cDNA ends) method, to obtain twocomplete long cDNA sequences (Hfzo1 and Hfzo2).

Hfzo1 had a total length of 2754 bp (SEQ ID NO: 1), and its ORF encodedHzo1 protein made up of 741 amino acid residues (SEQ ID NO: 2) andhaving a molecular mass of about 84 kD. Hfzo2 had a total length of 2754bp (SEQ ID NO: 3), and its ORF encoded Hzo1 protein made up of 769 aminoacid residues (SEQ ID NO: 4) and having a molecular mass of about 87 kD.

Both of these proteins were identified as a GTPase having transmembranedomains. Specifically, as to the Hfzo1, transmembrane domains were foundat the positions 595–607 and 614–628 in SEQ ID NO: 2, while as to theHfzo2, transmembrane domains were found at the positions 623–635 and642–656 in SEQ ID NO: 4. The GTPase active site of Hfzo1 was found atthe position 76–268 in SEQ ID NO: 2, while the GTPase active site ofHfzo2 was observed at the position 104–296 in SEQ ID NO: 4. In addition,Coiled Coil domains were observed at the positions 360–404 and 689–734in SEQ ID NO: 2 for Hfzo1, and at the positions 388–432 and 717–762 inSEQ ID NO: 4 for Hfzo2. Furthermore, these genes have alternativesplicing at their 5′ terminal exons, and in particular, Hfzo2 proteinhas a structure predictable as a mitochondrial target signal (position1–31 in SEQ ID NO: 4).

Example 2 Expression of Hfzo cDNA in HeLa Cell

Polynucleotides comprising base sequences constituting ORF regions ofSEQ ID NO: 1 and SEQ ID NO: 3 were individually inserted into expressionvectors (pCMV-SPORT) and coupled thereto, and the resultant recombinantvectors were introduced into HeLa cells, the HeLa cells were stainedwith rhodamine 123, and the condition of mitochondria was observed.

The result is shown in FIG. 1. In FIG. 1, the upper stage shows abright-field image and the lower stage shows a dark-field image. Fromthese images, it was demonstrated that mitochondria aggregate in thevicinity of nuclei in the HeLa cell into which either cDNA of Hfzo1 orcDNA of Hfzo2 has been introduced (middle lane and right lane,respectively), in contrast to the control (left lane) where reticulummitochondria are observed throughout the cytosol. Also,electromicroscope observation of HeLa cell into which Hfzo2 cDNA hasbeen introduced demonstrated partial induction of mitochondrial innermembrane.

From these results, it was confirmed that the Hfzo1 and Hfzo2 proteinsof the present invention have the function of promoting aggregation andfusion of mitochondria.

In addition, point mutation analysis with respect to the GTPase activesite demonstrated that aggregation of mitochondria depends on GTPaseactivity.

Example 3 Expression of Hfzo cDNA-GFP Fusion Protein in HeLa Cell

GFP was coupled to a carboxylic terminal of polynucleotides comprisingeither of the base sequences constituting ORF regions represented by SEQID NO: 1 and SEQ ID NO: 3, and the resultant polynucleotide was insertedand coupled into an expression vector (pCMV-SPORT: Life Technologies),to create a plasmid. The plasmid was co-expressed in a HeLa cell withpDs Red1-Mito plasmid (Clontech) encoding red fluorescent protein havinga mitochondrial target signal. Using a confocal laser scanningmicroscope, mitochondria were observed by red fluorescence and Hfzo1-GFPand Hfzo2-GFP fusion proteins by green fluorescence.

The results are as shown in FIG. 2. In FIG. 2, the first (in aleft-to-right order) lane shows bright-field image, the second laneshows Hfzo-GFP fusion protein observation image (green fluorescence),the third lane shows mitochondria observation image (red fluorescence)and the fourth lane shows a simultaneous observation image ofmitochondria and Hfzo-GFP fusion protein. The upper stage shows the HeLacell into which Hfzo1-GFP cDNA has been introduced, and the middle stageshows the HeLa cell into which Hfzo2-GFP cDNA has been introduced. Thelower stage shows an image of mitochondria portion of the HeLa cell intowhich Hfzo2-GFP cDNA has been introduced, observed at strongmagnification. As is apparent from FIG. 2, it was confirmed that both ofthe Hfzo proteins locate on mitochondrial membrane, and specificallylocate at the site where mitochondria aggregate and contact with eachother.

Example 4 Preparation of Recombinant Hfzo2 Protein

Host Escherichia coli BL21 was transformed with an expression vector(pQE and pGEX) into which a polynucleotide comprising the base sequenceconstituting the ORF region of SEQ ID NO: 3 had been inserted. Thebacterial cells were cultured for 5 hours at 37° C. in LB medium, addedwith IPTG in a final concentration of 0.4 mM, and cultured for another2.5 hours at 37° C. Bacterial cells were separated by centrifugation,dissolved in a lysis buffer (50 mM Tris HCl (pH7.5), 1 mM EDTA-1% TritonX-100, 0.2% SDS, 0.2 mM PMSF), frozen at −80° C. and melted, and thencrushed by ultrasonication. The crushed substances were centrifuged, andHfzo2 protein was separated and purified from the supernatant.

Example 5 Preparation of Proteoliposome

10 mg of soybean phospholipid was dissolved in 1 mL of chloroform, and50 μl of the solution was evaporated to give a pellet. This pellet washydrated with 100 μL of 75 mM KCl and 10 mM phosphate buffer (pH 7.0),and freezing-melting cycle was repeated three times. Thereafter, thesolution was fractionated through a 0.2 μm filter to prepare a liposome.

Then this liposome and the purified Hfzo2 protein obtained in Example 4were mixed in equal amounts, followed by freezing and melting, toprepare a proteoliposome of mono-double layer membrane.

INDUSTRIAL APPLICABILITY

According to the present invention, novel human proteins which promoteaggregation and fusion of mitochondria, polynucleotides encoding suchproteins, antibodies against such proteins, and proteoliposomescontaining such proteins are provided. Human proteins are useful forclarifying causes of mitochondrial diseases as well as for developingpreventive and therapeutic methods thereof. Furthermore, antibodies andprobes derived from genes encoding such proteins are potentially usefulmaterial for diagnosis of condition of mitochondria in particulardiseases. Furthermore, proteoliposome provides measures for specifictransfer of foreign genes or drugs targeted toward mitochondria.

1. An isolated and purified human mitochondrial protein comprising theamino acid sequence of SEQ ID NO:
 2. 2. A polynucleotide encoding theprotein of claim
 1. 3. The polynucleotide of claim 2, which comprisesthe base sequence for the protein translation region in SEQ ID NO:
 1. 4.An expression vector, which expresses the polynucleotide of claim 3 inan in vitro translating system or in a host cell.
 5. A transformantcontaining an expression vector which produces the human mitochonddalprotein of claim 1, said expression vector expressing a polynucleotidecomprising the base sequence for the protein translation region in SEQID NO: 1.